Trypanosoma cruzi glycoprotein vaccine for inducing immunity to Chagas&#39; disease

ABSTRACT

A novel, glycoprotein antigen obtained from T. cruzi organisms can be used in vaccines for inducing immunity in humans to Chagas&#39; disease. The glycoprotein is extracted by treating trypanosomes with a detergent and separating it from the cell debris and other proteinaceous material by affinity chromatography using lectin with affinity for glucose, mannose or galactose.

The present invention relates to vaccines against Chagas disease. Inparticular it relates to antigenic material enriched in glycoproteinsobtained from epimastigotes of T. cruzi and a process for producing thesame.

Chagas' disease is endemic in several countries of Central and SouthAmerica, especially Mexico, Brazil, Chile, Argentina and Venezuela. Itis caused by the osmotrophic protozoan, Trypanosoma cruzi, which istransmitted by the common, blood sucking, reduviid bugs betweenvertebrate hosts such as man, domestic pets including cats and dogs, andwild mammals. Chagas' disease is especially dangerous because there areno satisfactory prophylactic or curative agents available and because anindividual, once he has contracted the disease remains infected forlife.

Because of the present lack of effective chemotherapy, several attemptshave been made to develop vaccines for immunisation against Chagas'disease.

T. cruzi are known to exist in a number of different morphologicalstages depending upon their environment. In the infected mammal T. cruzioccur as trypomastigotes and amastigotes while in the insect vector orin axenic culture in vitro, they may be induced to change into theepimastigote stage. This latter stage is relatively easily grown and hasbeen utilised for many years in the production of antigen forimmunological and diagnostic studies.

Several methods have been employed to prepare antigens from T. cruzi.Antigens prepared using mechanical rupture of T. cruzi organisms byfreezethawing, trituration with or without beads, and sonicationpartially protect mice from acute trypanasomiasis (Johnson P., and Neal,R. A., Nature (1963), 200, 83; Goble F. C. et al., J. Parasitol, (1964),50 (Supple), 19; Seneca, H. et al. Nature, (1966), 209, 309-310;

However, generally a decrease in parasitaemia but little increase insurvival was observed. Homogenisation of epimastigotes of T. cruzi, at140 atm. pressure in a nitrogen atmosphere with precise temperaturecontrol, preserved the subcellular structures intact and permitted theirisolation (Segura, E. L. et al., J. Protozool, 21, 571-574). Subsequentto this work, Gonzalez-Cappa S. M. et al. (J. Parasitology 1976, 62,130) and Segura, E. L. et al. (Ibid. 131) disrupted epimastigotes of T.cruzi by nitrogen cavitation and fractionated the homogenate bycentrifugation. They discovered that protective activity was closelyrelated with the flagellar fraction which afforded 90% survival of mice.The other fractions showed some activity, but was not as efficient asthe flagellar fraction.

Unfortunately it has been noticed that many of these antigens,especially the flagellar fractions, although providing a degree ofimmunity to Chagas' disease, may cause symptons of the disease whichhave been associated with auto-immune effects (Texeira A. R. L. et al.Am. J. Pathol., (1975), 80, 163-180). For this reason is probable thatsimple sub-cellular fractions are unsuitable for use in vaccines.

Seneca has described a polysaccharide-containing antigen, isolated fromT. cruzi organisms, (Nature, 209, 309, (1966) and known as"chagastoxin". Recent work has shown chagastoxin to be a complex of"lipopeptidophosphoglycan" and several other components (Colli et al, J.Protozool, 21, 575, (1974); FEBS Letters, 52, 188, (1975); Biochem,Biophys. Acta, 444, 85, (1976); Eur. J. Biochem., 74, 263, (1977);Gottlieb, J. Immunol., 119, 465, (1977)) but these are now known not tobe protective against experimental Chagas' disease (Colli et al., Rev.Inst. Med. Trop. Sao Paulo, 20, 246, (1978) and Gottlieb, oralcommunication during the International Congress of Protozoology, NewYork, 1977).

A soluble polysaccharide antigen was isolated by Goncalves and Yamaha(J. Trop. Med. Hyg., 72, 39, (1969)) and Bergendi et al. (Exp.Parasitol. 28, 258 (1970)) but there is no indication that this isprotective against Chagas' disease.

It has now been found that protein fractions obtained from culturedepimastigotes and containing certain glycoproteins are useful ininducing immunity to Chagas' disease.

It appears that the glycoprotein fraction produced by the methoddescribed below is free from the crossreacting determinants that arethought to cause the auto-immune phenomena found in infectedindividuals. The presence of such determinants in epimastigotes andfractions thereof may be demonstrated by testing for interaction of theepimastigote or a fraction thereof with components which can recogniseheart tissue in the antisera of patients suffering from chronic Chagas'disease.

According to the present invention there is provided an antigen whichcontains glycoproteins of molecular weight within the range from 6 to9.5×10⁴ obtained from T. cruzi organisms, the glycoproteins beingsubstantially insoluble in water and capable of interreacting withlectins which have an affinity for glucose, mannose or galactose, theantigen being substantially free from non-proteinaceous matter.

The molecular weight of the above-mentioned proteins can be estimatedconveniently using polyacrylamide gel electrophoresis techniques(Laemmli, U. K., Nature (Lond.), (1970), 227, 680-685).

The glycoprotein of the present invention is effectively insoluble inwater. However, when isolated according to the process disclosed below,the glycoprotein fraction may acceptably be associated with up to 5% byweight of water-soluble material of non-proteinaceous nature.

Lectins are proteinaceous materials capable of interacting with certainsugar moieties, e.g. glucose units, while having no appreciableinteraction with other sugars. They may therefore be used fordistinguishing which types of sugar are present in a glycoside such as aglycoprotein. The glycoproteins of the present invention are capable ofinteracting with those lectins which are known to have an affinity forglucose, mannose or galactose moieties.

The antigenic glycoprotein is a relativey fragile molecule, and issusceptable to breakdown by proteases also found in T. cruzi.Accordingly, care must be exercised in the extraction and purificationof the antigen.

According to a second aspect of the present invention there is provideda process for extracting and purifying the above-mentioned antigencomprising of the steps of

(a) harvesting trypanosmes from a culture;

(b) solubilising the antigen and

(c) removing substantially all non-glycoproteinaceous proteins byaffinity chromatography using lectins with affinity for glucose, mannoseor galactose.

The antigen is found in all stages of T. cruzi, however it is convenientto produce it from epimastigotes as this stage can most easily becultured. Epimastigotes are advantageously grown in Bone and Parentsmedium from trypomastigotes contained in a blood sample taken from aninfected animal. The epimastigotes are maintained by serial passage inthat medium with 5% rabbit serum, pencillin (200 units/ml) andstreptomycin (100 units/ml), but other culture media may be used such asLit (modified) medium at 28° C. (Gutteridge et al. J. Protozoology; 21;5127; (1969). Trypomastigotes and amastigotes may be obtained frominfected animals or cultured in vitro, e.g. using the method of Stohlmanet al Arch. Microbiol; 9; 301-311; (1973).

Advantageously, amastigotes may be grown in vitro at 37° C. in thepresence of the mouse muscle sarcoma cell line 52 in Dulbecco's modifiedEagles medium containing 10% foetal calf serum, penicillin andstreptomycin.

In step (a) the trypanosomes may conveniently be harvested by low speedcentrifugation, preferably below 8000 g min since greater forces tend tocause the break-down of the antigen, preferably at between 200 g and 600g for 10 minutes and most preferably at about 400 g for 10 minutes. Thecells are then washed with phosphate buffered (pH 7.2) saline (PBS).

Conditions for centrifugation are expressed in units of g min, that isthe force applied multiplied by the duration of the force. Thus 4000 gmin encompasses conditions such as 400 g for 10 minutes as well as 2000g for 2 minutes and so on within reasonable limits. Certain conditionswithin the generality have been found particularly advantageous andthese are expressed specifically as for instance 400 g for 10 minutes.

In step (b) the antigen may be solubilised by the addition of detergentto a suspension of the cells in PBS. No specific time or temperature isrequired for solubilisation, but 5 minutes at 0° C. has been foundconvenient for a suspension of 5×10⁸ cells/ml.

Detergents may be of the ionic or non-ionic type. However use of theionic detergents may promote the formation of DNA gels and non-ionicdetergents are therefore more convenient. Examples of non-ionicdetergents are Nonidet P 40, Triton X-100 and Brij 99. (Registered TradeMarks, manufactured by Shell, Rohn and Hass Co. and I.C.I. respectively)and a polyoxyethylene (12) tridecyl ether detergent, e.g. Renex 30(Registered Trade Mark, Manufactured by Honeywell Atlas Limited,) whichis the most preferred detergent. Detergent is added to a finalconcentration of between 1 and 5% preferably about 2% v/v.

Other undesirable proteins are removed in step (c) for instance byaffinity chromatography using lectins (see, for instance Hayman M. J.and Crumpton M. J., Biochem and Biophys Research Comms, 47, 923, (1972))which possess affinity for glucose, mannose or galactose. ConcanavalinA. or lectins from Ricinis communis are suitable for this purpose.Lectin obtained from Lens culinaris with affinity for glucose andmannose is most preferred.

The lectins are prepared for use in affinity chromatography columns bylinking the protein to an inert support such as Sepharose 4b activatedwith cyanogen bromide. After application of the antigen from step (b) tothe affinity column, non-glycoproteinaceous material is washed throughthe column with an eluant solution containing detergent. The eluant mayadvantageously contain buffers and salts, conveniently 0.15 M sodiumchloride and 0.01 M tris-hydrochloride pH 7.4 buffer are used, but thesemay be omitted or used at other concentrations, and other buffers andsalts may be used provided that extremes of pH and high saltconcentrations are avoided. The desired glycoprotein is eluted from thecolumn by addition of a sugar to the eluant, to a concentration of 1% to4% preferably 2% w/v of eluant is used, sugars such as glucose ormannose or their derivatives may be employed, however it is preferredthat methyl mannoside is used. Alternatively the glycoprotein may berecovered by denaturing the lectin, e.g. using concentrated sodiumdodecyl sulphate solution.

The purification process may be improved by the optional step ofremoving cell debris before affinity chromatography. It has been foundconvenient to use centrifugation at about 450,000 g min. Preferablyabout 15,000 g for 30 minutes although somewhat higher or lower speedsproviding forces down to about 3000 g for 30 minutes may also beemployed. Alternatively the cell debris can be removed by filtration.

The yield of antigen may be enhanced by the addition, to the cellsuspension, of protease inhibitors, either before step (b) or at thesame time as the addition of detergent. While these inhibitors may beomitted on small scale production of the antigen, on larger scales, itis more important that proteolysis is prevented otherwise most of theantigen will be destroyed during the prolonged purification procedure.Protease inhibitors also protect the lectin affinity columns, allowingre-use of the columns which would otherwise be damaged by proteases.

There are three classes of protease inhibitors; those which inhibitproteases in general, those which are specific for serine proteases andthose which are specific for sulphydryl proteases. General inhibitorsare exemplified by aprotinin, metal chelating agents such asethylenediamine tetracetate (EDTA) and metal ions such as mercury orzinc ions. Inhibitors of serine proteases include tosyl-L-lysinechloromethyl keytone hydrochloride (TLCK), phenylmethylsulphonicfluoride, and di-isopropyl fluorophosphate. Iodoacetamide, iodoaceticacid or p-chloromercuri benzoate are examples of sulphydryl proteasesinhibitors. There are many other inhibitors of proteases which are knownin the literature, however it is found convenient to use aprotinin, TLCKand iodoacetamide, and these are the most preferred inhibitors.Concentrations of aprotinin of 2 to 10, preferably about 5 units/ml, ofTLCK of 0.5 to 2 mM, preferably 1 mM and of iodoacetamide of 5 to 20 mMpreferably about 10 mM are particuarly recomended.

When used to asses the purity of the antigen polyacrylamide gelelectrophoresis shows that the glycoprotein eluted from the affinitycolumn comprises a plurality of components of estimated molecular weightbetween 6 and 9.5×10⁴. The most preferred glycoprotein has a molecularweight of about 9×10⁴ and the other components of the antigen arefragments of this glycoprotein. The antigen could be further purified byknown techniques, however the effort and concomitant loss of antigen isnormally unnecessary and wasteful as these glycoprotein fragments retaintheir antigenicity.

The antigen may be used in the form eluted from the affinity column, ormay be further processed, such as by precipitation with an organicsolvent such as an alcohol, e.g. ethanol and then resuspended as aparticulate, or solubilised by addition of detergent solution. Suchfurther steps, whilst producing more highly purified antigen also causea reduction in the overall yield of the antigen.

The concentration of solutions of the antigen may be estimated, forinstance, by spectrophotometry. Conveniently the absorption at 280 nmmay be measured and the concentration of the antigen is then determinedusing the extinction coefficient of 1.2 for a solution of 1 mg ofprotein per ml.

The antigen described above may be incorporated into a vaccine forinducing immunity to Chagas' disease in susceptible hosts such asmammals, including humans, at risk to be infected by T. cruzi. For thispurpose the antigen may be presented in association with apharmaceutically acceptable carrier.

According to the present invention in a further aspect there is provideda vaccine for inducing immunity to Chagas' disease which comprises anantigen as hereinbefore defined in association with a pharmaceuticallyacceptable carrier.

Pharmaceutically acceptable carriers, in this instance, are liquid mediasuitable for use as vehicles to introduce the antigen into the patient.An example of such a carrier is saline solution. The antigen may besuspended as a solid in the carrier, or it may be solubilised by theaddition of pharmaceutically acceptable detergent.

The vaccine may also comprise an adjuvant for stimulating the immuneresponse and thereby enhancing the effect of the vaccine. Convenientadjuvants for use in the present invention include Freunds completeadjuvant and more particularly, saponin Corynebacterium parvum(coparvax) and aluminum hydroxide or a mixture of these or other knownadjuvants.

Conveniently the vaccines are formulated to contain a finalconcentration of antigen in the range of from 0.2 to 5, preferably 0.5to 2, most preferably 1, mg/ml. After formulation the vaccine may beincorporated into a sterile container which is then sealed and stored ata low temperature, for example 4° C., or may be freeze dried.

In order to induce immunity in humans to Chagas' disease one or moredoses of the vaccine, formulated as described above, may beadministered. It is recommended that each dose is 0.1 to 2 ml preferably0.2 to 1 ml, most preferably 0.5 ml of vaccine.

The present invention in a further aspect provides a method for inducingimmunity to Chagas' disease in susceptible hosts, comprising theadministration of an effective amount of a vaccine, as hereinbeforedefined, to the host.

An effective amount of the vaccine is that quantity which is sufficientto induce, in the host animal, immunity to Chagas' disease.

The vaccines of the present invention are desireably administered bysubcutaneous or intramuscular injection although the intravenous routemay be employed. The treatment may consist of a single dose of vaccineor a plurality of doses over a period of time. An advantageous treatmentschedule requires administration of two doses of vaccine with aninterval of 7 to 56, preferably 14 days between doses. If longerprotection is required, booster doses may be administered after longerintervals, for instance, annually.

The following Examples serve to illustrate the invention but are notintended to limit it in any way.

EXAMPLE 1 Extraction and purification of antigen

Trypomastigotes were obtained in a blood sample from an infected animaland cultured in Bone and Parent's liquid medium (Bone, G. J. et al.; J.Gen. Microbiol.; 31, 261-266; (1963)) with 5% rabbit serum, penicillin(to 200 units/ml) and streptomycin (to 100 units/ml) added whereuponthey developed into epimastigotes which were maintained by serialpassage in Bone and Parents medium.

Cultures were established with 1×10⁷ epimastigotes/ml and incubated at26° C. for 4-7 days in glass bottles containing the medium (1000 ml)with gentle agitation. At the end of the culture period the mediumcontained ca. 2×10⁸ epimastigotes/ml. These were separated from thesupernatant by centrifugation at 400 g for 10 minutes and washed withphosphate-buffered (pH 7.2) saline (PBS). The pellet was resuspended inPBS. (5×10⁸ epimastigotes/ml) and detergent (Renex 30) (to 2% v/v),aprotinin (to 5 units/ml), tosyl-L-lysine chloromethyl ketonehydrochloride (TLCK) (to 1 mM) and iodoacetamide (to 10 mM) were addedto the suspension. The suspension was maintained at 0° C. for fiveminutes then centrifuged (15,000 g for 30 min) to remove the celldebris.

The supernatant (200 ml containing the product of 2×10¹¹ cells) wasapplied to the top of an affinity chromatography column (2.5×10 cmcontaining Lens culinaris, glucose- and mannose-binding lectin supportedon cyanogen bromide activated Sepharose 4b at 10 mg protein/mlSepharose) and pre-washed extensively with detergent solution (1% v/vRenex 30, 10 column volumes) containing sodium chloride (0.15 M) andtris-hydrochloride buffer (0.01 M, pH7.4). The antigen was then elutedusing methyl mannoside solution (2% w/v) containing detergent (1% v/vRenex 30), salt (0.15 M sodium chloride) and buffer (0.01 M trishydrochloride pH7.4) until no further glycoprotein was eluted (as shownby absorption at 280 n m). The glycoprotein appeared in the first 20 mlof methyl mannoside solution to run off the column.

The eluate was collected and the antigen precipitated by adding ethanol(3 volumes) and allowing it to stand at -20° C. for 48 hours. Theprecipitate was recovered by centrifugation at 2500 g for 15 min,affording 10 mg of the glycoprotein.

The purity of the glycoprotein fraction was assayed by polyacrylamidegel electrophoresis.

EXAMPLE 2 Preparation of a vaccine

The precipitate of Example 1 was resuspended in saline solution (0.9%w/v) and emulsified with an equal volume of Freunds complete adjuvant.The vaccine represented a 0.1% w/v suspension of glycoprotein.

EXAMPLE 3 Protection Studies

Groups of 10, C₅₇ BL mice were immunised intraperitoneally and/orsubcutaneously with 100 μl doses of the vaccine of Example 2 at days 0and 14. Control mice were either untreated or received Freunds completeadjuvant alone.

The mice were challenged on day 28 with 5×10³ blood streamtrypomastigotes of T. cruzi. All control mice died (mean survival time21 days, peak parasitaemia 3×10⁷ parasites/ml of blood) whilst theimmunised mice were all still alive on day 61 when the experiment wasterminated. In these latter groups peak parasitaemia, at day 21, was5×10⁵ parasites/ml of blood, and there were no microscopicallydetectable parasites after day 38.

I claim:
 1. A vaccine for inducing immunity to Chagas' diseasecomprising an antigen obtained from T. Cruzi organisms comprisingglycoprotein of molecular weight from about 6×10⁴ to about 9.5×10⁴, saidglycoprotein being substantially insoluble in water and being capable ofinteracting with lectins which have an affinity for glucose, mannose orgalactose, said antigen being substantially free from non-proteinaceousmatter and a pharmaceutically acceptable carrier therefor.
 2. A vaccineaccording to claim 1 which further comprises an adjuvant for stimulatingthe immune response.
 3. A method for inducing immunity to Chagas'disease comprising the administration to a susceptible host, of aneffective amount of the vaccine of claim
 1. 4. A method according toclaim 3 comprising the administration of two doses of vaccine at aninterval of from about 7 to about 56 days.